Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official VSports app下载. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2010 Nov 5;285(45):34439-46.
doi: 10.1074/jbc.M110.152306. Epub 2010 Sep 7.

MicroRNA-223 regulates cyclin E activity by modulating expression of F-box and WD-40 domain protein 7

Yanfei Xu  1 , Tanushri Sengupta, Lokesh Kukreja, Alex C Minella
Affiliations

"V体育平台登录" MicroRNA-223 regulates cyclin E activity by modulating expression of F-box and WD-40 domain protein 7

Yanfei Xu et al. J Biol Chem. .

Abstract

F-box and WD-40 domain protein 7 (Fbw7) provides substrate specificity for the Skp1-Cullin1-F-box protein (SCF) ubiquitin ligase complex that targets multiple oncoproteins for degradation, including cyclin E, c-Myc, c-Jun, Notch, and mammalian target of rapamycin (mTOR). Fbw7 is a bona fide tumor suppressor, and loss-of-function mutations in FBXW7 have been identified in diverse human tumors. Although much is known about targets of the Fbw7 ubiquitin ligase pathway, relatively little is known about the regulation of Fbw7 expression. We identified a panel of candidate microRNA regulators of Fbw7 expression within a study of gene expression alterations in primary erythroblasts obtained from cyclin E(T74A T393A) knock-in mice, which have markedly dysregulated cyclin E expression. We found that overexpression of miR-223, in particular, significantly reduces FBXW7 mRNA levels, increases endogenous cyclin E protein and activity levels, and increases genomic instability. We next confirmed that miR-223 targets the FBXW7 3'-untranslated region VSports手机版. We then found that reduced miR-223 expression in primary mouse embryonic fibroblasts leads to increased Fbw7 expression and decreased cyclin E activity. Finally, we found that miR-223 expression is responsive to acute alterations in cyclin E regulation by the Fbw7 pathway. Together, our data indicate that miR-223 regulates Fbw7 expression and provide the first evidence that activity of the SCF(Fbw7) ubiquitin ligase can be modulated directly by the microRNA pathway. .

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Identification of candidate microRNA regulators of Fbw7 expression in cyclin ET74A T393A erythroblasts. A, schematic of screen for dysregulated microRNAs in sorted basophilic erythroblast cells of the indicated immunophenotypes, isolated from two adult, homozygous cyclin ET74A T393A mice and two wild-type, littermate controls. The screen utilized a multiplex RT-PCR-based assay allowing the expression of 518 unique microRNAs to be measured via card array format. B, down-regulated expression of four candidate microRNAs with predicted target sites within the FBXW7 3′-UTR was independently verified, using individual quantitative RT-PCR assays. Displayed is a representative comparison of microRNA expression in primary erythroblasts isolated from two wild-type (light bars) and cyclin ET74A T393A mice. Error bars indicate standard deviations with triplicate measurements. C, candidate microRNA regulators of Fbw7 expression were validated in a Dual-Luminescence reporter assay performed in K562 cells, utilizing a reporter construct containing the full-length 3′-UTR of FBXW7 downstream of the Renilla luciferase reading frame. Normalized Renilla luciferase activities for the four candidate microRNAs, transfected as pre-miRs, are shown as ratios to the normalized activity found with expression of a control miR sequence. Error bars indicate standard deviations with separate transfections.
FIGURE 2.
FIGURE 2.
Direct microRNA targeting of the FBXW7 3′-UTR region by miR-25 and miR-223. A, mutation of the predicted target site for miR-25 within nucleotides 285–291 in the FBXW7 3′-UTR abrogates miR-25-mediated repression of reporter activity but not miR-223-mediated repression. ctrl, control. B, mutation of the predicted target site for miR-223 within nucleotides 189–196 abrogates miR-223-mediated repression of reporter activity and not miR-25-mediated repression. C, expression of FBXW7 in pre-miR-transfected HCT116s was measured by quantitative real-time PCR, using a probe designed to detect all isoforms of Fbw7. Error bars indicating standard deviations and p values are displayed. D, HCT116 cells were transfected with the indicated pre-miRs and either empty vector (ctrl) or a reporter construct expressing Fbw7α, with N-terminal FLAG tag and full-length 3′-UTR. Fbw7 protein levels were then measured by anti-FLAG immunoblot.
FIGURE 3.
FIGURE 3.
MicroRNA-223 regulates cyclin E stability and activity. A, left panel, HCT116 cells were transfected to express the indicated microRNAs, and endogenous total and threonine 380-phosphoryated cyclin E (T380-P Cyclin E) protein and activity levels were assayed by immunoblotting and H1 kinase assays. In this experiment, to demonstrate internal consistency with our transfection results, miR-27a and miR-27b were expressed separately; these are encoded by distinct pre-miRs, which are processed to yield miRs that differ only by a single nucleotide. Endogenous cyclin E kinase activities were quantified using a PhosphorImager and are indicated, and p values were calculated from three independent experiments (Student's t test, n.s., not significant). Right panel, expression of cyclin E1 in pre-miR-transfected HCT116s was measured using quantitative real-time PCR. B, parental and Fbw7-null HCT116 cells were transfected and endogenous cyclin E protein and activity measured as in A. We verified equivalent transfection efficiencies of 90% in both parental and Fbw7-null HCT116s. C, HCT116 cells were transduced with GFP-expressing retroviral vectors encoding control sequence or miR-223. Fluorescence-activated cell sorts were performed followed by metabolic labeling and endogenous cyclin E half-life determination. Both primary data and quantitation of S35-labeled cyclin E protein decay with calculated half-lives are shown. D, micronucleation was measured in HCT116 cells fixed 72 h following transfection with the indicated pre-miRs. Micronuclei were compared among the cell populations after counting 1000 cells/sample in two separate experiments and expressed as ratios to normal nuclei. Error bars indicating standard deviations and p values are displayed.
FIGURE 4.
FIGURE 4.
Endogenous miR-223 regulates Fbw7 expression in primary mouse embryonic fibroblasts. A, comparison of the indicated microRNA levels in HCT116 cells versus early passage MEFs using real-time PCR. B, levels of endogenous miR-25, miR-223, and Fbxw7 mRNA (all isoforms) were measured in synchronized, passage 1 MEFs. Calculated p values are displayed for the expression differences between G0 and G1 for both miR-25 and miR-223 in triplicate assays from two experiments. n.s., not statistically significant. C, left, levels of endogenous miR-25 and miR-223 were measured following transfection of primary MEFs with antagomirs (a-m). Right, MEFs were transfected with the indicated antagomirs and the reporter construct expressing FLAG-Fbw7α. FLAG-Fbw7 signal was quantified and normalized using the Grb2 signal; expression of antagomirs targeting miR-25 and miR-223 resulted in 3.7- and 3.5-fold increases in FLAG-Fbw7 expression, respectively, when compared with control (ctrl) antagomir-expressing cells. NS, nonspecific protein in MEF extract that cross-reacts with the FLAG antibody. D, left, primary MEFs were transfected with the indicated antagomirs and GFP plasmid. Transfected cells were sorted via flow cytometry, and lysates were prepared from collected cells, immunoblotted as shown, and immunoprecipitated for cyclin E kinase activity. Right, MEFs were co-transfected with the indicated antagomirs and CD20-expresssing plasmid, enabling gating of transfected cells. Cells were fixed following CD20 surface marker staining, and cell cycle distributions were measured. Data displayed represent mean values from triplicate experiments with p values for decrease in S/G2-M-phase populations indicated. Error bars indicating standard deviations are displayed in all panels. n.s., not statistically significant.
FIGURE 5.
FIGURE 5.
MiR-223 expression levels are responsive to acute alterations in cyclin E expression associated with manipulation of the Fbw7 pathway. A, K562 cells were transduced with control or cyclin E (T380A)-expressing retrovirus. Left panel, RT-PCR analyses were performed to measure the abundance of the indicated microRNAs in cyclin E (T380A)-expressing cells, relative to control vector-transduced cells. Right panel, immunoblot analysis of transduced K562 cells for cyclin E and Grb2 (loading control). Statistical significance for expression differences was determined as for Fig. 4B. B, K562 cells were transduced with control or FLAG-Fbw7α-expressing retrovirus followed by similar microRNA RT-PCR and immunoblot analyses as in A. Error bars indicating standard deviations and p values are displayed. n.s., not statistically significant. C, schematic of proposed negative feedback loop connecting cyclin E levels to regulation of Fbw7 expression via miR-223.
See this image and copyright information in PMC

References (V体育2025版)

    1. Mao J. H., Kim I. J., Wu D., Climent J., Kang H. C., DelRosario R., Balmain A. (2008) Science 321, 1499–1502 - VSports手机版 - PMC - PubMed
    1. Oberg C., Li J., Pauley A., Wolf E., Gurney M., Lendahl U. (2001) J. Biol. Chem. 276, 35847–35853 - PubMed (VSports手机版)
    1. Nateri A. S., Riera-Sans L., Da Costa C., Behrens A. (2004) Science 303, 1374–1378 - PubMed
    1. Welcker M., Orian A., Jin J., Grim J. E., Harper J. W., Eisenman R. N., Clurman B. E. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 9085–9090 - PMC - PubMed
    1. Yada M., Hatakeyama S., Kamura T., Nishiyama M., Tsunematsu R., Imaki H., Ishida N., Okumura F., Nakayama K., Nakayama K. I. (2004) EMBO J. 23, 2116–2125 - PMC - PubMed

"VSports" Publication types

MeSH terms

Substances

LinkOut - more resources